Isolated/cloned human nt2 cell lines expressing serotonin and gaba

ABSTRACT

Human cells isolated and/or cloned from human NT2 cells for expressing serotonin (5HT) and gamma-aminobutyric acid (GABA) are disclosed. These cells can be used as cellular minipumps to release serotonin and/or GABA to treat various neurological diseases, conditions, or disorders, particularly neurodegenerative disorders and the consequences of brain and spinal cord injuries (pain/spasticity).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority on prior U.S. ProvisionalApplication Ser. No. 60/463,315, filed Apr. 17, 2003, and which isincorporated herein in its entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work leading to the present invention was supported by one or moregrants from the U.S. Government. The U.S. Government therefore hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention is directed to the creation of two novel celllines, the GABAergic hNT2.17 and serotonergic hNT2.19, synthesizing andreleasing the neurotransmitters GABA and serotonin (5HT), both derivedfrom the human NT2 cell line, and which are useful for the treatment ofpain and spasticity and other traumatic or neurodegenerative injuries tothe nervous system. These cells are stably transplanted into host rodentanimals in models of pain and spasticity.

Chronic pain and spasticity constitute some of the significant problemsfollowing spinal cord injuries (SCI), interfering with rehabilitationand daily activities, which in turn leads to significant complicationsin these patients. However, there are no effective, completely safetherapies for the reduction of these conditions (and other related toneural trauma and degeneration).

Oral baclofen, the GABA-B receptor agonist, has often been the drug ofchoice for spasticity (Reference 1) due to spinal cord trauma, andbaclofen and opioids have been used for the relief of chronic pain(Reference 2). However, oral baclofen is often ineffective at non-toxicdoses, because large doses are required to cross the blood-brain barrierand can lead to subsequent CNS effects (Reference 3), usually not welltolerated by spastic patients. Intrathecally administered baclofen iscurrently FDA-approved for those patients with spinal cord injury whoare refractory or can not tolerate oral baclofen, but spinally-deliveredbaclofen via programmable implantable electronic pumps is also fraughtwith problems, such as high costs, catheter twisting, infection at theimplant site, overdosing and the development of tolerance withincreasing doses of baclofen required for relief (References 4 and 5).Furthermore, the efficacy of baclofen pumps is mainly in terms of lowerextremity spasticity and the treatment has little effect on upperextremity spasticity. Intrathecal morphine has been used via electronicpump and has been prescribed for use in neuropathic pain, but often hascomplications of tolerance, overdose, and problems related to the pumpitself (References 6-8).

Transplants of primary cultured cells near the dorsal horn of the spinalcord that release peptides and neurotransmitters have offered a newdirection in the treatment of chronic pain (Reference 9). However,primary cells are difficult to obtain, non-homogeneous, and wouldrequire that each batch be tested before clinical use. Transplantationof immortalized cell lines genetically-modified to release neuroactiveantinociceptive peptides (Reference 10), inhibitory neurotransmitters(References 11 and 12) and neurotrophins (Reference 13) in chronic pain,and to upregulate inhibitory neurotransmitter synthesis (Reference 14)offers a renewable source of cells that can act as cellular minipumps,are able to respond to the microenvironment of the cord, and shouldreduce or eliminate side effects associated with the large doses ofpharmacologic agents required for centrally-acting pain-reducing agents.A naturally-immortalized human embryonal carcinoma cell line,NTera2cl.D/I (NT2), differentiates irreversibly into severalmorphologically and phenotypically distinct cell types, which includeterminally differentiated postmitotoc CNS neurons (Reference 15).Successive re-plating of retinoic acid-treated NT2 cells, in thepresence of growth inhibitors, results in the isolation of purifiedhuman neurons (Reference 16), that have been extensively characterizedand tested in vivo in a number of animal models of traumatic injury andneurodegenerative disease (Reference 17). The potential application ofNT2 neurons in cell transplantation therapy for CNS disorders, and theiruse as vehicles for delivering exogenous proteins into the human brainfor gene therapy, has been recently demonstrated (Reference 18). SuchNT2 neurons are being currently used in Phase-II clinical trials for thetreatment of stroke, and have been approved by FDA for such trials(References 19 and 20). However, such NT2 cells contain a variety ofneural phenotypes, and would provide a plethora of neuroactivesubstances if used for the specific treatment of problems such as painor SCI-associated spasticity.

U.S. Pat. Nos. 5,082,670, 5,175,103, 5,792,900, 6,162,428, 6,214,334,and 6,254,865, are directed to the use of the parental human NT2 cellline for a variety of disorders, but none describe the creation or useof individual cell lines derived from the NT2 or NT2/D cell lines.

In order to ameliorate various neurological diseases, conditions, ordisorders, the present invention creates two novel human neural celllines, subcloned and derived from the human NT2 cell line, where eachsubcloned cell line synthesizes and secretes bioactive agents, such GABAor serotonin (5HT). Transplants of these secreting human neural celllines are believed to reduce consequences of spinal trauma aftertransplant in or near the spinal cord.

OBJECTS AND SUMMARY OF THE INVENTION

The principal object of the present invention is to provide an isolatedand/or cloned human cell and/or cell line which expresses a bioagent,such as serotonin (5HT) or gamma-aminobutyric acid (GABA).

Another object of the present invention is to provide an isolated and/orcloned human cell and/or cell line expressing serotonin (5HT) orgamma-aminobutyric acid (GABA) which can be used to treat a neurologicaldisease, condition, or disorder, such as pain, spasticity, epilepsy,depression, mobility disorder, sensory disorder, Parkinson's, orAlzheimer's disease.

Yet another object of the present invention is to provide an isolatedand/or cloned human cell and/or cell line expressing serotonin (5HT) orgamma-aminobutyric acid (GABA) that releases specific bioagents toreverse the damage to the nervous system, which can be safely used fortransplant into or near the brain or spinal cord after injury or indisease states.

An additional object of the present invention is the use of clonal humanhNT2 neuronal cell lines as cellular minipumps to release serotonin(5HT) and/or gamma-aminobutyric acid (GABA) to treat neurodegenerativedisorders and consequences of brain and spinal cord injuries.

In summary, the main object of the present invention is to provide anisolated and/or cloned human cell and/or cell line that expressesserotonin (5HT) or gamma-aminobutyric acid (GABA). The cells can be usedto treat various neurological diseases, conditions and/or disorders,such as pain, spasticity, epilepsy, depression, mobility disorder,sensory disorder, Parkinson's, and Alzheimer's, and/or to reverse thedamage to the nervous system.

At least one of the above-noted objects is met, in part, by the presentinvention, which in one aspect includes a cell for expressing serotonin(5HT) isolated from a human NT2 cell line.

Another aspect of the present invention includes a cell for expressinggamma-aminobutyric acid (GABA) isolated from a human NT2 cell line.

Another aspect of the present invention includes a cell for expressingserotonin (5HT) cloned from a cell obtained from a human NT2 cell line.

Another aspect of the present invention includes a cell for expressinggamma-aminobutyric acid (GABA) cloned from a cell obtained from a humanNT2 cell line.

Another aspect of the present invention includes treating a neurologicaldisease, condition, or disorder by administering to a subject in needthereof a suitable amount of serotonin (5HT) expressing hNT2 cells.

Another aspect of the present invention includes treating a neurologicaldisease, condition, or disorder by administering to a subject in needthereof a suitable amount of gamma-aminobutyric acid (GABA) expressinghNT2 cells.

Another aspect of the present invention includes a composition fortreating a neurological disease, condition, or disorder, which includesa cell for expressing a bioagent selected from the group consisting ofserotonin (5HT), and yaminobutyric acid (GABA), and a suitable carrier,wherein the cell is isolated from a human NT2 cell line.

Another aspect of the present invention includes a composition fortreating a neurological disease, condition, or disorder, which includesa cell for expressing a bioagent selected from the group consisting ofserotonin (5HT), and yaminobutyric acid (GABA), and a suitable carrier,wherein the cell is cloned from a cell obtained from a human NT2 cellline.

Another aspect of the present invention includes a method of producing amammal useful for studying a neurological disease, condition, ordisorder by transplanting a suitable amount of serotonin expressinghuman NT2 cells into or near a mammal's spinal cord, brain, orperipheral nervous system.

Another aspect of the present invention includes a method of producing amammal useful for studying a neurological disease, condition, ordisorder by transplanting a suitable amount of gamma-aminobutyric acid(GABA) expressing human NT2 cells into a mammal's spinal cord, brain, orperipheral nervous system.

Another aspect of the present invention includes a cell transplantmaterial, which includes serotonin expressing human NT2 cells.

Another aspect of the present invention includes a cell transplantmaterial, which includes gamma-aminobutyric acid (GABA) expressing humanNT2 cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, novel features and advantages of thepresent invention will become apparent from the following detaileddescription of the preferred embodiment(s) invention, as illustrated inthe drawings, in which:

FIG. 1A is a phase-contrast photomicrograph of differentiated (one week)hNT2.19 cells;

FIG. 1B is a phase-contrast photomicrograph of differentiated (two week)hNT2.19 cells;

FIG. 1C is a photomicrograph of a sample of two-week differentiatedhNT2.19 cells stained for an antibody directed against serotonin (5HT);

FIG. 1D is a photomicrograph of another sample of hNT2.19 cells stainedin the same manner as the cells of FIG. 1C;

FIG. 2A is a phase-contrast photomicrograph of differentiated (one week)hNT2.17 cells;

FIG. 2B is a phase-contrast photomicrograph of differentiated (two week)hNT2.17 cells;

FIG. 2C is a photomicrograph of a sample of two-week differentiatedhNT2.17 cells stained for an antibody directed against GABA;

FIG. 2D is a photomicrograph of another sample of hNT2.17 cells stainedin the-same manner as the cells of FIG. 2C;

FIG. 2E is a photomicrograph of hNT2.17 cells, differentiated two-weeksand stained with the nuclear marker DAPI to illustrate viable cells;

FIG. 2F is a photomicrograph of the same culture of cells shown in FIG.2E, co-labeled for the neuronal marker NeuN;

FIG. 2G is a photomicrograph of another sample of two-weekdifferentiated culture of hNT2.17 cells stained with DAPI to

FIG. 2H is a photomicrograph of the same culture of cells shown in FIG.2G, co-labeled for the human marker NuMA;

FIG. 3 is a bar chart showing GABA synthesis in two-week differentiatedhNT2.17 and hNT2.19 cell lines in vitro;

FIGS. 4A-4B are bar charts showing GABA release, stimulated by low (FIG.4A) or high concentrations (FIG. 4B) of K+ in hNT2.17 and hNT2.19 celllines in vitro;

FIG. 5A is a graphical illustration showing reversal of thermalhyperalgesia (tail-flick) in rats by hNT2.17 and hNT2.19 cells, wherecells were grafted after complete sacral transection (S2);

FIG. 5B is a bar chart illustrating spasticity (flexor tone) followingS2 lesion and transplant of either hNT2.17 or hNT2.19 cells;

FIG. 5C is a bar chart illustrating spasticity (extensor tone) followingS2 lesion and transplant of either hNT2.17 or hNT2.19 cells;

FIG. 5D is a bar chart illustrating spasticity (stretch reflex)following S2 lesion and transplant of either hNT2.17 or hNT2.19 cells;

FIG. 5E is a bar chart illustrating spasticity (flexor spasm) followingS2 lesion and transplant of either hNT2.17 or hNT2.19 cells;

FIG. 5F is a bar chart illustrating spasticity (cutaneous reflex)following S2 lesion and transplant of either hNT2.17 or hNT2.19 cells;

FIG. 6A is a photomicrograph illustrating surviving and viable (DAPIpositive) hNT2.19 grafted cells on the spinal cord after excitotoxicSCI;

FIG. 6B is a photomicrograph illustrating surviving and viable (DAPIpositive) hNT2.19 grafted cells co-labeled for the human marker NuMAafter excitotoxic SCI;

FIG. 6C is a photomicrograph illustrating surviving and viable (DAPIpositive) hNT2.17 grafted cells on the spinal cord after excitotoxicSCI;

FIG. 6D is a photomicrograph illustrating surviving and viable (DAPIpositive) hNT2.17 grafted cells co-labeled for the human marker NuMAafter excitotoxic SCI;

FIG. 7A is a graphical illustration showing reduction/reversal oftactile hypersensitivity in a model of excitotoxic SCI pain; and

FIG. 7B is a graphical illustration showing reduction/reversal ofthermal hyperalgesia in a model of excitotoxic SCI pain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) OF THE INVENTION

The availability of an inexhaustible supply of human cells which canmake specific biologic agents to reverse the damage to the nervoussystem that could be safely used for transplant into or near the brainor spinal cord after injury or in disease states would be very useful totreat a variety of problems such as pain, spasticity, epilepsy,immobility or related problems. Ideally such cell sources should not bederived from human embryos or cadavers, and should not form tumors ifplaced in the patient, but would still function and survive for longperiods. Such cell transplants would not be required to replace missingcells, but would function as cellular minipumps, releasing beneficialmolecules near damaged tissue so that some of the damaged tissue andhence, human function, could be repaired or improved.

The present invention is directed to deriving some of these clinicallyuseful cells by isolating unique colonies of daughter cells from anaturally-immortal human neuronal cell line called hNT2, so that thedaughter colonies are different from each other and the original parenthNT2 cell type, a process called subcloning. However, these new daughtercell lines retain the property of being naturally-immortal, when theyare grown in the absence of retinoic acid (RA). Is this condition, theymultiply indefinitely, allowing them to be frozen, stored, and restartedat any time in culture. If the cells are treated with RA for a fewweeks, they become human neurons, no longer capable of division. Suchcells can be safely transplanted into the potential patient, with nodanger of tumor formation, since they become human neurons irreversibly,which no longer divide and multiply. In addition, since they are uniquederivative cell lines, they have characteristic features, such as theability to make and secrete useful agents, such as the neurotransmittersserotonin (5HT) and gamma-aminobutyric acid (GABA).

The ability of transplants of these unique hNT2 cell lines to relievechronic pain and spasticity was tested in animal models after spinalcord injury (SCI), and it is expected that they would be beneficial forthese conditions. Transplant of cell lines that secrete 5HT or GABA thatcould be used in the human conditions, such as the consequences of SCIwhere chronic pain and spasticity are important problems clinically,would make them a desirable new tool for the SCI population. However,such subcloned cell lines, derived from the parental hNT2 neuronal cellline have great potential for cell therapy for the treatment of avariety of traumatic and neurodegenerative diseases in humans. Examplesof what these and other subcloned NT2 cell lines could make includeGABA, 5HT, catecholamines, opioids, as well as many other peptides oragents unknown at this time.

The present invention is therefore directed to isolating novel andspecific, well-characterized cell lines from the hNT2 parent line byconventional subcloning methods, and testing the use of individual novelNT2 cell lines for the treatment of traumatic consequences, such as painand spasticity. However, such cell line transplants could also be usedto treat a variety of neural trauma and neurodegenerative problems,including epilepsy, depression, mobility and sensory disorders,including Parkinson's and Alzheimer's disease.

The basis of the proposed efficacy of this invention is that release ofneuroactive agents, such as GABA or 5HT, from such cell transplants willprovide a low, local dose of agent in the area of neural substrate whereneeded, without a broad systemic effect, and such cells, sinceimmortalized and grown in vitro can be well-characterized and tested forhomogeneity and safety for human use.

Subcloning and Characterization of NT2 GABA and 5HT Neuronal Cell Lines

Conventional methods for serial dilution and subcloning with cloningrings are used to isolate proliferating and rapidly-growing colonies ofthe parental hNT2 cells, that form cell lines, in vitro (Reference 21).A more rapid cell-aggregation method (Reference 22) is used, describedbelow, for the differentiation of proliferating NT2 cells that onlyrequires 14 days of retinoic acid (RA) treatment, to characterize eachNT2 clonal line for a GABA or 5HT phenotype. Each clonal line isinitially stained for GABA or 5HT production with immunohistochemicalmethods (Reference 12) using commercially-available antibodies directedagainst GABA or 5HT. GABA- or 5HT-containing NT2 cell lines aredouble-labeled with the neuronal marker, neuronal nuclear marker (NeuN)(Reference 23), or the human nuclear matrix antigen, NuMA (Reference 24)to insure the phenotype is both GABAergic or 5HTergic and neuronal. Thecontent and release of the neurotransmitter GABA or 5HT from candidateGABA or 5HT NT2 cell lines is evaluated by standard high performanceliquid chromatography (HPLC) methods, described below, and is used tocharacterize other neurotransmitter cell lines (References 12 and 25).Cell lines which synthesize and release greater than 100 pmole GABA or5HT/10⁶ cells/min are used for further characterization and transplantexperiments. Some of the NT2 neuronal cell lines which do not synthesizeor secrete GABA or 5HT and do not stain positive for these agents byantibody methods, but survive well in vitro are used as thenegative-control NT2 cell line for further transplant work in models ofpain and spasticity after SCI.

Rapid Aggregation Method for Differentiation and Identification of NT2Clones

This method was adapted from an earlier published aggregation method forNT2 cell cultures (Reference 22). Confluent proliferating NT2 cells,expanded in non-tissue culture 100 mm plates, are treated for two weekswith 10 micromolar all-trans retinoic acid (RA), in high-glucose DMEMmedia, made pH 8.0 with 15 mM HEPES buffer. Remaining cells are replatedin poly-L-lysine/laminin-(PLS/lam)coated tissue culture plates (20ug/ml), and the high-glucose DMEM supplemented with 2 mM Glutamine, pH7.4. Twenty-four hours after replating, cytosine-D-arabinofuranoside(araC), 1 micromolar, and uridine, 10 micromolar, are added to the mediafor seven days. Differentiation is continued for one or two weeks afterthe removal of the mitogen inhibitors on PLS/lam-coated plastic slides.Individual clonal lines are examined for antibody staining andcharacterization for GABA or 5HT and NuMA or NeuN and other markers.

Immunohistochemistry Methods: In Vitro and In Vivo

Staining for GABA or 5HT in NT2 cell lines is done in a conventionalmanner (Reference 12). The specific GABA or 5HT signal are examined inboth proliferating and differentiated cell lines, as well as intransplant grafts, in models of pain and spasticity. Any suspected GABAor 5HT cell lines are also examined for GABA or 5HT content and releaseby HPLC (below) to insure that any suspected GABA or 5HT synthesis andrelease from the cells is authentic.

Methods for NeuN staining have been previously published (Reference 23).The human-specific NuMA has been examined in parental hNT2 grafts invivo (Reference 24). NeuN and NuMA are examined in differentiated clonalcultures to insure that clonal cell lines are human neurons. Typicalmethods for in vitro GABA, NeuN, and NuMA staining are as follows. Fixcells with Lana's (4% paraformaldehyde plus 10% picric acid, pH 7.4) at4° C. for ten minutes. Rinse and permeabilize cells with 0.4% TritonX-100 in PBS. Incubate ten minutes at room temperature. Incubate withprimary antibody for 12 hrs at 4° C. For serotonin (5HT): Rabbitantisera raised against 5HT (Immunostar), 1:1,000/PBS. For GABA: Guineapig antisera raised against GABA (Protos Biotech Corp.), 1:250 PBS. ForNeuN: Mouse anti-neuronal Nuclei monoclonal antibody (Chemicon) 1mg/ml., 1:100 PBS. For NuMA: Monoclonal NuMA (Ab-2) (Oncogene) 100ug/ml., 1:100 PBS. Rinse and incubate with secondary antibody for 2 hrs.Typical secondary antibodies include: For GABA: Alexa Fluor 488 Goatanti-guinea pig (Molecular Probes), 1:150 PBS. For NeuN and NuMA: Alexafluor 488 Goat anti-mouse (Molecular Probes), 1:150 PBS. For brightfieldimages, ABC Elite kits (Vector), with VIP substrate, and appropriatesecondary for each species of primary antibody are used.

HPLC GABA in NT2 Cell Lines

Either GABA content is measured in water-lysed cells, or GABA release ismeasured in buffer containing normal (2.95 mM) or high (100 mM) KClincubated for 15 min with cultures of two-week differentiated cells. TheHPLC system consists of a solvent-delivery pump (Waters 510 Pump), anautosampler (Waters 717 plus Autosampler) and an electrochemicaldetector (ESA Coulochem II; Electrode: ESA Model 5011 Analytic Cell;Guard Cell: Model 5020). Elution is carried out at room temperature witha reversed-phase column (3 uM, C18, 80×4.6, HR-80, ESA) and a mobilephase of 0.1 M Sodium Acetate (pH=5)-acetonitrile (73:27, v/v) at a flowrate of 0.6 ml/min. To an OPA solution (2 mg of o-Phthaldialdehyde (OPA)in 0.2 ml Methanol), 0.8 ml of 0.1 M Borax Buffer, pH 10.4 and 5 μl of2-mercaptoethanol (2MCE) are added. 4 min before the injection on thecolumn, to prepare the OPA reagent. The OPA reagent and sample are mixed(1:4) and incubated at room temperature in the autosampler beforeinjection. After injection, the GABA peak appearance time is about 5 minin 27% Ace Mobile Phase. Determination of 5HT synthesis will followsimilar published methods (Reference 25).

Animal Model of Spasticity

A sacral transection for the development of spasticity has beendeveloped in a rat model (Reference 26). Complete sacral (S2) spinaltransection only affected the tail musculature, and otherwise wasminimally disruptive to normal functions, not interferring with bowel,bladder, or hindlimb locomotor function. After spinal transection,initially the tail musculature was paralyzed for two weeks, followed byincreasing hypertonia, hyperreflexia, and clonus that developed overweeks and remains permanent in tail function, easily assessed in theawake rat. Muscle stretch or cutaneous stimulation of the tail producedmuscle spasms and marked increases in muscle tone, measured with forceor EMG recordings (Reference 26). Spontaneous or reflex induced flexorand extensor spasms are readily seen in the unconstrained tail. The tailand areas surrounding the tail, including the skin and hair developthermal hyperalgesia and tactile allodynia, suggesting a variety ofsensory disturbances, including the development of chronic pain,features that often accompany spasticity at- or below-level spinalinjury in humans (Reference 27). These characteristic motor (andsensory) responses develop during 4 distinct time periods (stage 1-4,acute through chronic, day 1 to >60 days) and surgical rats are comparedto uninjured animals. The following rating system is used to evaluateanimals: 0, minimal activity (flacid); +, normal activity; ++, increasedactivity; and, +++, maximal activity. To test for thermal hyperalgesia,a tail-flick test is used (Reference 28), with an automated Hargreavesdevice to determine the latency of response to the noxious thermalstimulus. All spasticity and pain behaviors are examined before andafter surgeries until at least 60 days after S2 spinal lesion, with celltransplants placed two weeks after SCI.

Animal Model of Excitotoxic SCI and Chronic Pain

A recently developed model of excitotoxic SCI (Reference 29) is nowbeing used by many groups to study the mechanisms and behaviorsassociated with chronic pain and spinal injury (References 30-36). Thismodel not only allows for quantitative assessment of behavioralhypersensitivity after injury but, with focused spinal microinjectionsof the excitotoxic agent, also permits an investigation of the cellularmechanisms in the cord that might be associated with the onset of thatpain. As well, this excitotoxic SCI pain model has been used to evaluatethe effects of cell transplantation of primary adrenal chromaffin tissueto reverse the chronic behavioral allodynia and hyperalgesia (Reference36) that chemical lesioning of the dorsal horn pain processing centersproduces. The model makes use of intraspinal injection of the glutamatereceptor agonist, quisqualic acid (QUIS) just above the lumbar segmentswhich control sensory function in the hindlimbs, which leads topredictable and quantifiable temporal profile of pain behaviors, withoutthe complications of a loss in motor systems, paralysis, or loss ofbowel and bladder function (Reference 29).

Intraspinal injection of quisqualic acid (QUIS) produces injury withpathological characteristics similar to those associated with ischemicand traumatic SCI (References 35 and 37). The pathological changes afterQUIS include neuronal loss, demyelination, cavitation, glial responses,perivascular changes, breakdown of blood-brain barrier and inflammation(Reference 38). In addition, significant, evoked mechanical allodyniaand thermal hyperalgesia have been shown to be important components.Each of these behaviors is indicative of altered sensory function and/orpain, similar to that reported after SCI (Reference 39). After spinaltransplantation of primary adrenal tissue following QUIS injections,pain related behaviors, including the progression of spontaneousexcessive grooming behavior and hypersensitivity to mechanical stimuliwere significantly reduced (Reference 36). These transplantation resultsin a pain-related model of SCI suggests that similar human celltransplants might be a meaningful therapeutic strategy for the largeproportion of SCI patients that report intractable chronic pain afterSCI (Reference 40).

Allodynia is the response, measured as a change in threshold, to anormally non-painful stimuli, such as cold (0-4° C.) or tactilestimulation. Hyperalgesia is the response measured as a change inthreshold to a painful stimuli, such as heat or pressure. The thermalhyperalgesia response to heat using an automated Hargreaves device(References 28 and 41) requires that a constant intensity, radiant heatsource be aimed at the midplantar area of an animals paw and the latencyof response time from initial heat source activation until pawwithdrawal is recorded with an automatic timer. Mechanical allodynia isthe measure of foot withdrawal in response to normally innocuousmechanical stimuli using a graded series of von Frey hairs (Reference42). Each graded hair represents an increasing force in grams applied tothe extremity. Our laboratory now utilizes an automated von frey devicefor mechanical allodynia measurement. These behavioral measures ofallodynia and hyperalgesia are the most commonly used measures for painin a variety of animal and human models, including neuropathic painafter spinal injections of quisqualic acid (References 19, 36, and 40).

Test Results from Human Cloned Cell Lines EXAMPLE 1 Serotonergic NT2Cell Line in Vitro—a Unique Serotonin hNT2 Cell Line (FIGS. 1A-D)

The hNT2.19 serotonergic cell line, was subcloned by serial dilution andtreated for two weeks with retinoic acid and mitotic inhibitors. Theywere further differentiated for two weeks before an antibody stain forserotonin (5HT). The 5HT NT2.19 cells, have a very large nucleus, aregenerally multipolar, with short neurites and stain brightly for 5HT.FIG. 1A is a phase-contrast photomicrograph of differentiated (one week)hNT2.19 cells; FIG. 1B is the same cells differentiated two weeks inculture. FIG. 1C is a sample of 2 week differentiated hNT2.19 cellsstained for an antibody directed against serotonin (5HT); FIG. 1D isanother photomicrograph of another hNT2.19 cell similarly stained.

EXAMPLE 2 The GABAergic NT2 Cell Line In Vitro—a Unique Neuronal GABAhNT2 Cell Line (FIGS. 2A-H)

Another cloned hNT2 cell line, hNT2.17, which is positive for GABA wassubcloned by serial dilution and treated for two weeks with retinoicacid and mitotic inhibitors. They were further differentiated for twoweeks before an antibody stain for GABA. The GABA NT2.17 cell line hassmall nuclei, extensive multipolar or bipolar neurites and stainsbrightly for GABA (FIGS. 2C and D). Other cultures of hNT2.17,differentiated for 2 weeks, were also stained for the neuronal marker,NeuN (FIG. 2F), and the human nuclear matrix antigen (NuMA) marker invitro (FIG. 2H). The counterstain DAPI, identifies viable, survivingcell grafts. These markers, especially human NuMA, can be used tospecifically identify hNT2 grafts in vivo after transplant in the rat.FIG. 2A is a phase-contrast photomicrograph of differentiated (one week)hNT2.17 cells; FIG. 2B is the same hNT2.17, cells differentiated twoweeks in culture. FIG. 2C is a sample of 2 week differentiated hNT2.17cells stained for an antibody directed against GABA; FIG. 2D is anotherphotomicrograph of another hNT2.17 cell similarly stained for GABA; FIG.2E is a photomicrograph of hNT2.17 cells, differentiated 2 weeks andstained with DAPI to illustrate viable cells; FIG. 2F is the sameculture co-labeled for the neuronal marker NeuN. FIG. 2G is another 2week differentiated culture of hNT2.17 cells again stained with DAPI toillustrate viability; FIG. 2H is the same culture co-labeled for thehuman marker NuMA.

EXAMPLE 3 GABA Content (Synthesis) in hNT2.17 and hNT2.19 Cell Lines InVitro—Only hNT2.17 Cells Synthesize GABA (FIG. 3)

Both the hNT2.17 and hNT2.19 cell lines were differentiated, afterretinoic acid and mitotic inhibitor treatment, for two weeks in 6-wellsubstrate-coated plates before cell lysis and examination of cellcontent for authentic GABA synthesis by HPLC methods (described above).Only the hNT2.17 cell line had any significant GABA content, matchingthe immunohistochemical staining patterns seen above. The hNT2.19 cells,which had stained for 5HT, not GABA, had no significant GABA contentexamined by HPLC methods.

EXAMPLE 4 GABA Release in hNT2.17 and hNT2.19 Cell Lines In Vitro—OnlyhNT2.17 Cells Release GABA (FIGS. 4A-B)

As above, both the hNT2.17 and hNT2.19 cell lines were differentiated,after retinoic acid and mitotic inhibitor treatment, for two weeks in6-well substrate-coated plates before cells were exposed to normal (2.95mM; FIG. 4A) or high (100 mM; FIG. 4B) concentrations of KCl forpotassium (K+)-stimulated release for authentic GABA by HPLC methods.Only the hNT2.17 cell line had any significant GABA release, matchingthe immunohistochemical staining pattern seen above. The hNT2.19 cells,which had stained for 5HT, not GABA, had little detectable GABA releaseexamined by HPLC methods.

EXAMPLE 5 Transplant of hNT2.17 and hNT2.19 Cell Lines in a Model ofSpasticity: Pain and Spasticity Behaviors—only hNT2.17 Cells ReverseSpasticity Behaviors, but Both hNT2.17 and hNT2.19 Reverse ThermalHyperalgesia in the Affected Extremity After Complete Spinal Transection(FIGS. 5A-F)

Adult male Wistar Furth rats were given a sacral (S2) complete spinaltransection in a rat model of spasticity (Reference 26) and eitherexamined for spasticity behaviors without cell transplants, or givensubarachnoid transplants of hNT2.17 or hNT2.19 cells (10⁶cells/injection) which had been differentiated for two weeks in vitro.The transplant was done at two weeks (14 days) after the original S2lesion (surgery). All animals were examined for spasticity behaviors andthermal hyperalgesia (heat sesnsitivity) in the tail-tip andmusculature. In FIG. 5A, thermal hyperalgesia was measured weekly at thetail tip after the S2 lesion (day 0), and after the S2 lesion, followedby cell grafts at 2 weeks after S2 injury, until about 65 days after theSCI. Both hNT2.17 and hNT2.19 reversed the thermal hyperalgesia inducedby the S2 transection, since this is a measure of the development ofchronic pain and hypersensitivity to noxious thermal stimuli. However,when spasticity behaviors are examined following the S2 lesion (FIGS.5B-F), only the grafts of the GABA-secreting hNT2.17 cells tended toreverse/reduce the spasticity behaviors (listed on the top of eachgraph). Serotonin from the grafts of hNT2.19 cells had no effect onthese spasticity behaviors, and these cells function as the negativecontrol transplant for the GABA cells.

EXAMPLE 6 Transplant of hNT2.17 and hNT2.19 Cell Lines in a Model ofExcitotoxic SCI and Chronic Pain: Immunohistochemistry—both hNT2.17 andhNT2.19 Survive Greater than 6 Weeks after Transplant in a Model of SCI(FIGS. 6A-D)

Adult male Wistar Furth rats were spinally injected with an excitoxicagent, quisqualic acid in a rat model of SCI and chronic pain(References 29, 38, 43, and 44) and spinal cord sections were examinedat 60 days after QUIS for evidence of surviving hNT2.17 and hNT2.19 cellline grafts. Either hNT2.17 or hNT2.19 cells (10⁶ cells/injection),which had been differentiated for two weeks in vitro, were injected intothe subaracnoid space two weeks (14 days) after the QUIS lesion. Cellgraft sites were co-localized with DAPI and the human marker NuMA (FIGS.6A-D). There are many surviving hNT2.17 and hNT2.19 grafted cellsvisible on the pial surface, which stain for the human-specific markerNuMA at the end of the experiment, 60 days after QUIS and about 6 weeksafter cell transplant. FIG. 6A illustrates surviving and viable (DAPIpositive) hNT2.19 grafted cells on the cord; FIG. 6B the same graftedhNT2.19 cells are co-labeled for the human marker NuMA. FIG. 6Cillustrates surviving and viable (DAPI positive) hNT2.17 grafted cellson the cord; FIG. 6D the same grafted hNT2.17 cells are co-labeled forthe human marker NuMA.

EXAMPLE 7 Transplant of hNT2.17 and hNT2.19 Cell Lines in a Model ofExcitotoxic SCI and Chronic Pain: Sensory Behaviors—both hNT2.17 andhNT2.19 Cell Grafts Reduce/Reverse Thermal and Tactile Hypersensitivityin a Model of SCI Pain (FIGS. 7A-B)

Adult male Wistar Furth rats were spinally injected with an excitoxicagent, quisqualic acid in a rat model of SCI and chronic pain(References 29, 38, 43, and 44), animals are either left untreated orinjected with either hNT2.17 or hNT2.19 cells (10⁶ cells/injection) intothe subarchnoid space at two weeks (14 days) after QUIS. Animals weretested before the SCI (baseline) and twice a week following QUIS andcell grafts for hypersensitivity to tactile or thermal stimuli inhindpaws below the SCI. All animals were examined for chronic painbehaviors in the contralateral (con) and ipsilateral (ipsi) hindpaws.Both ipsilateral and contralateral hindpaws recovered near-normalsensory responses to tactile and thermal stimuli after grafting eitherthe GABAergic hNT2.17 or serotonergic hNT2.19 cells, compared to theQUIS injury alone (measure of behaviors described above). QUIS injurynegatively affects hindpaw responses bilaterally, but the ipsilateralhindpaw is most affected by the injection of quisqualic acid. Neitherhindpaw recovers normal tactile or thermal responses after QUIS alone by60 days after the injection.

A pharmaceutical composition including an isolated or cloned human cellline expressing serotonin (5HT) or gamma-aminobutyric acid (GABA), maybe prepared, in a conventional manner. In particular, a pharmaceuticalcomposition made in accordance with the present invention would includean isolated or cloned human cell expressing serotonin (5HT) orgamma-aminobutyric acid (GABA), in an amount sufficient to providetherapeutic and/or prophylactic benefit, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Compositions of the present invention may be formulated forany appropriate manner for administration, including, for example,nasal, intravenous or intramuscular administration, or nervous system ornon-nervous system transplantation. Appropriate dosages, duration andfrequency of administration would be determined by known factors, suchas the condition of the patient, the type and severity of the diseaseand the method of administration.

While this invention has been described as having preferred sequences,ranges, steps, materials, or designs, it is understood that it includesfurther modifications, variations, uses and/or adaptations thereoffollowing in general the principle of the invention, and including suchdepartures from the present disclosure as those come within the known orcustomary practice in the art to which the invention pertains, and asmay be applied to the central features hereinbeforesetforth, and fallwithin the scope of the invention and of the limits of the appendedclaims.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

REFERENCES

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1. A cell for expressing serotonin (5HT) isolated from a human NT2 cell line.
 2. A cell for expressing gamma-aminobutyric acid (GABA) isolated from a human NT2 cell line.
 3. A cell for expressing serotonin (5HT) cloned from a cell obtained from a human NT2 cell line.
 4. A cell for expressing gamma-aminobutyric acid (GABA) cloned from a cell obtained from a human NT2 cell line.
 5. A method of treating a neurological disease, condition, or disorder, comprising: administering to a subject in need thereof a suitable amount of serotonin (5HT) expressing hNT2 cells.
 6. The method of claim 5, wherein: the hNT2 cells are transplanted in or adjacent to the spinal cord of the subject.
 7. The method of claim 5, wherein: the hNT2 cells are transplanted in or adjacent to the brain of the subject.
 8. The method of claim 5, wherein: the hNT2 cells are transplanted at a location in or adjacent to the central or peripheral nervous system of the subject.
 9. A method of treating a neurological disease, condition, or disorder, comprising: administering to a subject in need thereof a suitable amount of gamma-aminobutyric acid (GABA) expressing hNT2 cells.
 10. The method of claim 9, wherein: the hNT2 cells are transplanted in or adjacent to the spinal cord of the subject.
 11. The method of claim 9, wherein: the hNT2 cells are transplanted in or adjacent to the brain of the subject.
 12. The method of claim 9, wherein: the hNT2 cells are transplanted at a location in or adjacent the central or peripheral nervous system of the subject.
 13. A composition for treating a neurological disease, condition, or disorder, comprising: a) a cell for expressing a bioagent selected from the group consisting of serotonin (5HT), and gamma-aminobutyric acid (GABA), the cell having been isolated from a human NT2 cell line; and b) a suitable carrier.
 14. The composition of claim 13, wherein: a) the neurological disease, condition, or disorder comprises one or more of pain, spasticity, epilepsy, depression, mobility disorder, sensory disorder, Parkinson's, or Alzheimer's disease.
 15. A composition for treating a neurological disease, condition, or disorder, comprising: a) a cell expressing a bioagent selected from the group consisting of serotonin (5HT), and gamma-aminobutyric acid (GABA), the cell having been cloned from a cell obtained from a human NT2 cell line; and b) a suitable carrier.
 16. The composition of claim 15, wherein: a) the neurological disease, condition, or disorder comprises one or more of pain, spasticity, epilepsy, depression, mobility disorder, sensory disorder, Parkinson's, or Alzheimer's disease.
 17. A method of producing a mammal useful for studying a neurological disease, condition, and/or disorder, comprising: transplanting a suitable amount of serotonin expressing human NT2 cells into or near a mammal's spinal cord, brain, or peripheral nervous system.
 18. The method of claim 17, wherein the mammal is a rodent.
 19. A rodent produced in accordance with the method of claim
 17. 20. A method of producing a mammal useful for studying a neurological disease, condition, or disorder, comprising: transplanting a suitable amount of gamma-aminobutyric acid (GABA) expressing human NT2 cells into or near a mammal's spinal cord, brain, or peripheral nervous system.
 21. The method of claim 20, wherein the mammal is a rodent.
 22. A rodent produced in accordance with the method of claim
 20. 23. A cell transplant material, comprising: a) serotonin expressing human NT2 cells.
 24. A cell transplant material, comprising: a) gamma-aminobutyric acid (GABA) expressing human NT2 cells. 